! 1! Summary The proposal is in response to PA-19-237: Novel RNAs in Virology (including HIV) and Immune Regulation: Basic Science and Therapeutic Discovery. HIV-1 (HIV) infects CD4 positive cells causing acquired immunodeficiency syndrome (AIDS). Many forms of cell death (apoptosis, necrosis, necroptosis, autophagy, pyroptosis, mitotic catastrophe, and others) have been shown to be involved in virus induced cell loss of directly infected and bystander cells. While current antiretroviral therapy (ART) now prevents CD4 decline and restores their numbers to nearly normal in most cases, a major unsolved problem is why some cells survive HIV infection rather than dying, persist with latent provirus. A fundamental understanding of mechanisms of cell death induction by HIV could provide the means to kill those cells that constitute a reservoir of reactivatable virus that mandates lifelong ART. Such cells have so far evaded death from experimental ?shock and kill? cure strategies. We have recently discovered a novel form of cell death that is a combination of almost all of the above-mentioned mechanisms implicated to date in cell death associated with untreated HIV infection. 6mer Seed Toxicity (6mer Seed Tox) is an RNA interference (RNAi) based mechanism that kills cells through toxic seeds that target reverse complementary seed matches in the 3'UTR of a large number of genes that are critical for the survival of all cells. Our recent data suggest that primary tissues are protected from 6mer Seed Tox by highly expressed miRNAs that do not carry a toxic seed and block access of the potentially toxic small RNAs to the RNA induced silencing complex (RISC) that mediates RNAi. Our new preliminary data demonstrate that HIV infection kills cells that lack these protective miRNAs much more efficiently and it kills cells less efficiently that cannot form a functional RISC to mediate RNAi. These data suggest that cell death induced by HIV involves the RNAi machinery. The first hypothesis of this proposal is that HIV kills infected cells by engaging the 6mer Seed Tox mechanism either by triggering the generation of cell-endogenous toxic sRNAs or by producing virus-encoded toxic sRNAs that enter the RISC. The second hypothesis is that HIV stresses infected cells in a way that causes downregulation of the miRNA biogenesis enzyme Dicer decreases maturation of protective miRNAs, sensitizing infected cells to toxic siRNAs that are generated independently of Dicer. These hypotheses will be studied in two aims: Specific Aim 1: Determine whether HIV-1 triggers 6mer Seed Tox in infected cells through the production of toxic viral or cellular sRNAs. Specific Aim 2: Determine whether modulation of protective miRNAs in either direction renders HIV-1 infected cells more or less susceptible to 6mer Seed Tox. Our work will establish whether HIV kills infected cells through 6mer seed toxicity. It may pave the way to advance current HIV eradication strategies by sensitizing cells to induction of 6mer Seed Tox after latency reversal and decreasing neuronal death in HAND.